Asthma is an inflammatory lung disease often triggered by inhalation of protein allergens. Because of the central role of T cells in asthma, as with other allergic diseases, prevention may lie in treatments that enhance tolerance mechanisms. Injection of allergens subcutaneously has been used for many years as a treatment, called specific-allergen immune therapy (SIT or AIT), with the goal of inducing long-term tolerance. However, although SIT/AIT can undoubtedly be efficacious in some patients, it is a lengthy, inefficient, and often ineffective treatment, warranting new approaches that may quickly induce a state of tolerance and one that is maintained over time. As the development of inducible regulatory T cells (iTreg) and the concomitant deletion of memory T cells may be key to tolerance, new insights into the molecular control of these cells may then reveal therapeutic targets that can be combined with allergen treatment. We have shown in a mouse model of asthma that inhalation of pure protein antigen induces two iTreg populations, Foxp3+LAP- and Foxp3-LAP+ CD4 cells, that both can suppress Th2-driven allergic lung inflammation and protect against developing subsequent lung disease. Moreover, we identified a lung antigen-presenting cell (APC) that may be responsible for inducing these iTreg. We have further shown that signals through two pattern recognition receptors (PRR), TLR4 and Nod2, expressed in the lung, directly antagonize development of the iTreg and instead favor the development of pathogenic Th2 cells. Most importantly, these PRR drive the lung response through two cytokines, TSLP and IL-25, and a costimulatory member of the TNFR superfamily OX40L. Whether allergens elicit the same molecular signature to oppose tolerance is unclear, but we have found that generation of airway iTreg is inhibited by two extracts that are used for SIT in asthmatics and contain allergens associated with asthma, from Dermatophagoides pteronyssinus, a house dust mite (HDM), and from Aspergillus fumigatus, a fungus. Tolerance in allergic and asthmatic individuals also has to counter activities of pre-existing memory T cells We have further identified OX40L and another TNF costimulatory family ligand, LIGHT, as strong regulators of memory T cells that drive their clonal expansion and survival. Our hypothesis is then that long-term and effective tolerance will only be achieved to allergens in previously sensitized individuals if several of these molecules, and cytokines that synergize with these molecules, are coordinately suppressed. This proposal will determine the molecular signature elicited by several common human allergens, and test whether manipulating these cytokines and costimulatory ligands, and APC that drive iTreg induction, will be key to promoting tolerance in sensitized individuals during allergen immunotherapy.